The impact of past Roman occupation on the composition of ectomycorrhizal (ECM) communities was analysed in 12 Roman settlements in an oak forest in Central France. At each Roman settlement, soils and ECM roots were sampled from two plots (600 m2 each), one plot close to the remains of the buildings (<100 m), supposed to be impacted by ancient Roman agriculture, and the second plot 250–500 m away from the remains of the buildings, supposed to be less intensively influenced by previous cultivation. Soils were analysed and ECM fungal taxa were identified by morphotyping and sequencing the rDNA ITS region. The soil properties were significantly affected by the past Roman occupation, in terms of nutrient availability, especially for P, N and Mg. The enhancement of soil nutrient levels by past Roman land-use had significantly modified alpha diversity and species composition of ECM communities. Among the 67 determined ECM morphotypes, 40 were shared by the occupied and non-occupied plots, 17were found only in the occupied plots and 10 only in the non-occupied plots. Six morphotypes were significantly more frequent near the antique remnants. Our study showed, for the first time, that ectomycorrhizal communities are impacted by previous Roman land-use, even after nearly two thousand years of forest state.